molecules
Article
Synthesis and Evaluation of Novel Nitric Oxide-Donating
Ligustrazine Derivatives as Potent Antiplatelet
Aggregation Agents
Han-Xu Li, Jian-Hui Tian
Citation: Li, H.-X.; Tian, J.-H.; Li,
H.-Y.; Wan, X.; Zou, Y. Synthesis and
Evaluation of Novel Nitric
Oxide-Donating Ligustrazine
Derivatives as Potent Antiplatelet
Aggregation Agents. Molecules 2023,
28, 3355. https://doi.org/10.3390/
molecules28083355
Academic Editor: Changxing Qi
Received: 8 March 2023
Revised: 8 April 2023
Accepted: 9 April 2023
Published: 11 April 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Molecules 2023, 28, 3355. https://doi.org/10.3390/molecules28083355
, Hua-Yu Li, Xin Wan and Yu Zou *
Institute of Pharmaceutical Process, Hubei Province Key Laboratory of Occupationl Hazard Identification and
Control, School of Medicine, Wuhan University of Science and Technology, Wuhan 430065, China;
lihanxu997@sina.com (H.-X.L.)
* Correspondence: zouyu@wust.edu.cn
Abstract: Antiplatelet aggregation agents have demonstrated clinical benefits in the treatment of is-
chemic stroke. In our study, a series of novel nitric oxide (NO)-donating ligustrazine derivatives were
designed and synthesized as antiplatelet aggregation agents. They were evaluated for the inhibitory
effect on 50 -diphosphate (ADP)-induced and arachidonic acid (AA)-induced platelet aggregation
in vitro. The results showed that compound 15d displayed the best activity in both ADP-induced
and AA-induced assays, and compound 14a also showed quite better activity than ligustrazine. The
preliminary structure-activity relationships of these novel NO-donating ligustrazine derivatives were
discussed. Moreover, these compounds were docked with the thromboxane A2 receptor to study
the structure-activity relationships. These results suggested that the novel NO-donating ligustrazine
derivatives 14a and 15d deserve further study as potent antiplatelet aggregation agents.
Keywords: ligustrazine; nitric oxide donors; antiplatelet aggregation agents; structure-activity
relationships
1. Introduction
Stroke is a highly devastating brain disease and the second leading cause of death
across the world. The annual number of cases and deaths due to stroke increased substan-
tially worldwide, thereby resulting in an increasing medical health burden [1]. Among the
strokes, ischemic stroke (IS) is a major type commonly caused by thrombus and cerebral
arterial stenosis [2]. Despite its difficult treatment, therapeutic approaches against platelet
aggregation are still considered a key component of the management of IS, particularly in
the secondary prevention of cerebrovascular disease [3,4].
Traditional Chinese medicine is an important source of active natural products, many
of which have good therapeutic effects on vascular embolism and tissue ischemia. Chuanx-
iong (Ligusticum chuanxiong Hort), a traditional Chinese herbal medicine with a long history
of use in China, was first documented in the ancient medical book ShenNong’s Herbal Classic
as having the effects of promoting blood circulation, dispelling blood stasis and alleviating
pain [5]. Moreover, ligustrazine (2,3,5,6-tetramethylpyrazine, TMP), originally a natural
product extracted from chuanxiong, is made as an injection that is widely used as a clinical
drug for occlusive cerebrovascular disease in China [6,7]. TMP has a short half-life in vivo
because it is easily metabolized and rapidly excreted, which results in its limited actuation
duration [8]. Therefore, a large number of ligustrazine derivatives were synthesized and
modified to improve the effects and pharmacokinetic properties in the past few decades.
On the other hand, nitric oxide (NO), a well-known gaseous messenger molecule,
plays an important role in regulating physiological functions in cerebrovascular systems [9].
In cardiovascular and cerebrovascular systems, NO, produced by both endothelial cells
and platelets, has important antithrombotic effects by decreasing platelet activation [10].
https://www.mdpi.com/journal/molecules
 On the other hand, nitric oxide (NO), a well-known gaseous messenger molecule,
plays an important role in regulating physiological functions in cerebrovascular systems
Molecules 2023, 28, 3355 2 of 14
[9]. In cardiovascular and cerebrovascular systems, NO, produced by both endothelial
cells and platelets, has important antithrombotic effects by decreasing platelet activation
[10]. It is difficult to control NO gas when directly used in vivo, while NO donors’ organic
It is difficult
nitrate esters to
cancontrol
releaseNO NOgas when
into the directly
body viaused in vivo,orwhile
enzymatic NO donors’
nonenzymatic organic
pathways.
nitrate esters
Therefore, can release
organic NO into
nitrate esters canthe bodytovia
be used enzymatic
produce or nonenzymatic
the pharmacological pathways.
effects of NO
Therefore,
in vivo, such organic nitrate esters
as inhibiting canaggregation
platelet be used to andproduce the pharmacological
thrombus effects of
formation. Furthermore,
NO in vivo,various
connecting such as NO
inhibiting
donorsplatelet aggregationand
to cardiovascular andcerebrovascular
thrombus formation. Furthermore,
disease drugs was
connecting various NO donors to cardiovascular and cerebrovascular
a great strategy for enhancing their therapeutic activities and/or mitigating their disease drugs was a
adverse
great strategy
effects [11–13]. for enhancing their therapeutic activities and/or mitigating their adverse
effects
As[11–13].
mentioned, ligustrazine derivatives could avoid being rapidly metabolized in
vivo Asandmentioned, ligustrazine
prolong their actuation derivatives
duration,could
whileavoid being rapidlyofmetabolized
the introduction NO donorsintovivothe
and prolong their actuation duration, while the introduction of NO donors
structure could enhance antiplatelet aggregation activity. Thus, we connected the NO do- to the structure
couldwith
nors enhance antiplatelet
different aggregation
ligustrazine activity.
derivatives Thus, we
via various connected
linkers the NOseveral
to generate donorsnovel
with
different ligustrazine derivatives via various linkers to generate several
NO-donating ligustrazine derivatives (Table 1). It was expected that these compounds novel NO-donating
ligustrazine
could exhibitderivatives (Table 1).aggregation
better antiplatelet It was expected that these
activities. compounds
Herein, we reportcould exhibit better
the synthesis and
antiplatelet aggregation activities. Herein, we report the synthesis
biological evaluation of the novel NO-donating ligustrazine derivatives as potentand biological evaluation
an-
of the novel NO-donating
tiplatelet aggregation agents. ligustrazine derivatives as potent antiplatelet aggregation agents.

Table 1. Chemical structures of compounds 14a–14e and 15a–15e.

Table 1. Chemical structures of compounds 14a–14e and 15a–15e.

Compounds
Compounds Substituted
SubstitutedPosition
Position Linker
Linker
1313 / / /
/
14a
14a o- o- m
m= = 22
14b
14b m-m- m
m= 2
14c
14c p-p- m == 22
m
14d o- m=4
14d o- m=4
14e o- m=6
14e
15a o- o- mn= = 26
15a
15b o-m- nn == 2
15c
15b m-p- nn == 22
15d
15c p-m- nn == 24
15e m- n=6
15d m- n=4
15e m- n=6
2. Results and Discussion
2.1.
2. Chemistry
Results and Discussion
In order to obtain the target compounds, two types of ligustrazine intermediates,
2.1. Chemistry
2-hydroxymethyl-3,5,6-trimethylpyrazine (3) and (E)-3-(3,5,6-trimethylpyrazin-2-yl) acrylic
In order
acid (6) to obtainCompound
were selected. the target compounds,
3 is an activetwo types ofofligustrazine
metabolite ligustrazineintermediates, 2-
with antiplatelet
hydroxymethyl-3,5,6-trimethylpyrazine (3) and (E)-3-(3,5,6-trimethylpyrazin-2-yl)
aggregation [14]. Compound 6 is an acrylic acid derivative that was initially synthesized acrylic
acid (6) were selected.
for improved solubilityCompound 3 is an
and developed active
more metabolite
bioactive of ligustrazine
analogs with compounds
[15]. Therefore, antiplatelet
aggregation [14]. Compound 6 is an acrylic acid derivative that was initially
3 and 6 were often used as active compound fragments for the synthesis of target active synthesized
for improved
compounds in solubility and
the previous developed
studies [16,17],more
as was bioactive
the caseanalogs [15]. Therefore, com-
in our study.
poundsWe 3first
andsynthesized
6 were oftenthe
used as active
simplest compound fragments
NO-donating ligustrazine forderivatives,
the synthesis 13,ofby
target
cou-
active6compounds
pling in the previous
with a NO donor, which was studies
used as[16,17], as was the
the reference case in our
compound study.
for the activity study
We compounds.
of other first synthesizedThe the simplest
syntheses of NO-donating
3, 6 and 13 areligustrazine
shown in Schemederivatives, 13, by cou-
1. Commercially
pling 6 with a NO donor, which was used as the reference compound
available ligustrazine hydrochloride was neutralized with sodium hydroxide to obtain for the activity study
of other compounds. The syntheses of 3, 6 and 13 are shown in Scheme
ligustrazine trihydrate. Ligustrazine trihydrate was treated with an acetic anhydride 30% 1. Commercially
available
hydrogenligustrazine hydrochloride
dioxide solution was neutralized
in the presence 90 ◦ C tohydroxide
withatsodium
of acetic acid to obtain
obtain ligustrazine
N-Oxide 1. Compound 1 underwent a Boekelheide reaction with acetic anhydride at 130 ◦ C
to yield 2, which was hydrolyzed to 3. The total yield was 33.7%. Compound 6 was
smoothly synthesized from 3 via oxidation, the Wittig–Horner reaction and the hydroly-
sis reaction [18], with a total yield of 71.6%. Compound 7 was obtained via a reaction of
2-bromoethanol with AgNO3 in acetonitrile, followed by condensation with 6 to provide 13.
 ligustrazine trihydrate.
ligustrazine trihydrate. Ligustrazine
Ligustrazine trihydrate
trihydrate was
was treated
treated with
with an
an acetic
acetic anhydride
anhydride 30%
30%
hydrogen dioxide solution in the presence of acetic acid at 90 °C to obtain ligustrazine
hydrogen dioxide solution in the presence of acetic acid at 90 °C to obtain ligustrazine N- N-
Oxide 1. Compound 1 underwent a Boekelheide reaction with acetic anhydride
Oxide 1. Compound 1 underwent a Boekelheide reaction with acetic anhydride at 130 °C at 130 °C
to yield 2, which was hydrolyzed to 3. The total yield was 33.7%.
to yield 2, which was hydrolyzed to 3. The total yield was 33.7%. Compound 6 was Compound 6 was
smoothly synthesized
smoothly synthesized from
from 33 via
via oxidation,
oxidation, the
the Wittig–Horner
Wittig–Horner reaction
reaction and
and the
the hydrolysis
hydrolysis
Molecules 2023, 28, 3355 reaction [18], with a total yield of 71.6%. Compound 7 was obtained via a reaction 3of
of 14
2-
reaction [18], with a total yield of 71.6%. Compound 7 was obtained via a reaction of 2-
bromoethanol with AgNO 3 in acetonitrile, followed by condensation with 6 to provide 13.
bromoethanol with AgNO3 in acetonitrile, followed by condensation with 6 to provide 13.

Scheme 1. 1. Syntheses
1. Synthesesofof
Syntheses ofcompounds
compounds3,3,
compounds 3,6 and
66 and
and 13.
13.13. Reagents
Reagents and
andand conditions:
conditions: (a) 30%H
(a) 30%H 2O2, AcOH,◦90
Scheme Reagents conditions: (a) 30%H 2 O22, O 2, AcOH,
AcOH, 90 90C,
°C, 4 h, 69%; (b) Ac 2O, 130
◦ °C, 2.5 h, 38–52%; (c) 20%NaOH, rt, 12 h, 85–92%; (d) MnO 2, EtOH, reflux,
4°C,h, 469%;
h, 69%;
(b) Ac(b)2 Ac O, 130
O, 2130 C, °C, 2.538–52%;
2.5 h, h, 38–52%; (c) 20%NaOH,
(c) 20%NaOH, rt,h,1285–92%;
rt, 12 h, 85–92%; (d) MnO
(d) MnO 2 , 2, EtOH,
EtOH, reflux,
reflux, 6 h,
66 h,
h, 95%;
95%; (e)
(e) toluene,
toluene, NaH,
NaH, triethyl
triethyl phosphonoacetate,
phosphonoacetate, rt, rt, under
under dark,
dark, 8–10
8–10 h,
h, 80%;
80%; (f) ① KOH/H
(f) ① KOH/H22O, O,
95%; (e) toluene, NaH, triethyl phosphonoacetate, rt, under dark, 8–10 h, 80%; (f)
1 KOH/H O, 16 h,
16 h,
16 ② HCl/H
h, ② HCl/H22O, O, 97%;
97%; (g)
(g) AgNO
AgNO33,, CH
CH33CN,
CN, 7070
◦
°C, under
°C, under dark,
dark, 33 h,h, 78%;
78%; (h)
(h) 7,
7, DCC, DMAP,2CH
DCC, DMAP, CH22Cl
Cl22,,
2 HCl/H2 O, 97%; (g) AgNO3 , CH3 CN, 70 C, under dark, 3 h, 78%; (h) 7, DCC, DMAP, CH2 Cl2 ,
rt, 47%.
rt, 47%.
rt, 47%.
Derivatives 14a–14e
Derivatives 14a–14e were
were synthesized
synthesized fromfrom aa series
series of
of hydroxybenzoic
hydroxybenzoic acid deriva- deriva-
Derivatives 14a–14e were synthesized from a series of hydroxybenzoic acid acid deriva-
tives (Scheme
tives (Scheme
(Scheme2). 2). O-hydroxybenzoic
2).O-hydroxybenzoic
O-hydroxybenzoicacid acid was
acidwas treated
wastreated
treated with
with 1,2-dibromoethane
1,2-dibromoethane to obtain
to obtain
tives with 1,2-dibromoethane to obtain 8a,
8a, followed
8a, followed by condensation
by condensation with 6 to provide 9a. The nitrate ester of 14a was obtained
followed by condensation with with 6 to provide
6 to provide 9a. The9a.nitrate
The nitrate
ester ofester
14a of
was 14a was obtained
obtained from a
from aa reaction
from reaction of of 10a with
with AgNO
AgNO33 in in acetonitrile.
acetonitrile. The
The preparation
preparation of of 14b–14e
14b–14e followed
followed
reaction of 10a with10aAgNO3 in acetonitrile. The preparation of 14b–14e followed the same
the same
the same route.
route. Further,
Further, 15a–15e
15a–15e were
were synthesized
synthesized fromfrom aa series
series of
of hydroxybenzaldehyde
hydroxybenzaldehyde
route. Further, 15a–15e were synthesized from a series of hydroxybenzaldehyde deriva-
derivatives
derivatives [19]. First, hydroxybenzaldehyde
[19].hydroxybenzaldehyde
First, hydroxybenzaldehyde reacted with the corresponding brominated
tives [19]. First, reactedreacted
with thewith the corresponding
corresponding brominatedbrominated
alcohol
alcohol to
alcohol to give
give 10a–e.
10a–e. Then,
Then, compounds
compounds 11a–e 11a–e were
were converted
converted to to 12a–e
12a–e by by nitration
nitration and
and
to give 10a–e. Then, compounds 11a–e were converted to 12a–e by nitration and oxidation.
oxidation.
oxidation.
Finally, Finally, condensation
Finally, condensation
condensation of 12a–e
of 12a–e
of 12a–e with with
with
3 in the 3 in the
3 in the of
presence presence of N,N′-dicyclohexylcar-
N,N0 -dicyclohexylcarbodimide
presence of N,N′-dicyclohexylcar-
bodimide (DCC) and 4-dimethylaminopyridine (DMAP)
(DCC) and 4-dimethylaminopyridine (DMAP) formed 15a–15e.15a–15e.
bodimide (DCC) and 4-dimethylaminopyridine (DMAP) formed
formed 15a–15e.
Except for Except
Except for com-
for
compounds com-
pounds
15a–15c, 15a–15c,
pounds 15a–15c,
separated separated
separated by thin-layer
by thin-layer
by thin-layer chromatography,
chromatography,
chromatography, the otherthe other compounds
the compounds
other compounds were
were were pu-
pu-
purified
rified
rified
by by
column column
by column chromatography.
chromatography.
chromatography. All the structures
All structures
All the the structures
were were
were identified
identified
identified by1 1H NMR,1313C NMR
bybyHHNMR,
1 NMR, CC NMR
13 NMR
and HRMS.
and HRMS.

Scheme
Scheme 2. 2. Syntheses
Syntheses of of compounds
compounds 14a–14e and
compounds 14a–14e
14a–14e and 15a–15e. Reagents
and 15a–15e.
15a–15e. Reagents and
Reagents and conditions:
and conditions: (a)
conditions: (a) Br(CH
(a) Br(CH222))m
Br(CH Br,
mBr,
mBr,
Et
Et N, acetone, reflux, 5 h, 29–60%; (b) 6, DCC, DMAP, CH Cl , rt, 70–93%; (c) AgNO , CH CN, ◦70
Et33N, acetone,
3
acetone, reflux,
reflux,55h,h,29–60%;
29–60%;(b)(b)
6, 6,
DCC,DCC,DMAP,
DMAP, CHCH
2 Cl22Cl
2 , rt,
2, 70–93%;
2
rt, 70–93%;(c) AgNO 3 , CH
(c) AgNO 3
3, 3 CN,
CH 3 70 70
3CN, C,
°C,
underunder
dark,
°C, under dark, 3 h, 37–64%;
3 h,337–64%;
dark, h, 37–64%; (d)
(d) (d) Br(CH
Br(CH
Br(CH 2 )
2 )n2OH,
n
)nOH,OH, K 2
K2KCO CO
2CO
3 , acetone,
3 , 3acetone,
reflux,
, acetone,reflux, 6 h, 29–67%;
reflux,66h,h,29–67%;
29–67%;(e)(e) Fuming
(e)Fuming
Fuming HNO HNO333,,
HNO
AcOH, Ac
AcOH, Ac22O,
O, –5 °C to
◦ C to rt,
rt, 22 h,
h, 60–66%;
60–66%; (f)(f) KMnO
KMnO44,,, acetone,
acetone, reflux,
reflux, 222 h,
h, 82–90%; (g) 3, DCC, DMAP,
AcOH, 2 O, –5
–5 °C to rt, 4 acetone, reflux, h, 82–90%;
82–90%; (g)
(g) 3,
3, DCC,
DCC, DMAP,
DMAP,
CH 2Cl2, rt, 43–54%.
CH22Cl22,, rt,
CH rt, 43–54%.
43–54%.

2.2. In Vitro Antiplatelet Aggregation

We evaluated the inhibitory effects of all the target compounds on platelet aggregation
induced by adenosine 50 -diphosphate (ADP) (10 µmol·L−1 ) and arachidonic acid (AA)
(1 mmol·L−1 ) in rabbit platelet plasma using the Born turbidimetric method. The results,
shown in Figure 1, showed that compound 15d displayed the best activity in the ADP-
induced and AA-induced assays (IC50 = 0.7347 mM and 0.5565 mM, respectively), both of
which were more active than TMP under the same conditions.
 We evaluated the inhibitory effects of all the target compounds on platelet aggrega-
tion induced by adenosine 5′-diphosphate (ADP) (10 µmol·L−1) and arachidonic acid (AA)
(1 mmol·L−1) in rabbit platelet plasma using the Born turbidimetric method. The results,
shown in Figure 1, showed that compound 15d displayed the best activity in the ADP-
Molecules 2023, 28, 3355 induced and AA-induced assays (IC50 = 0.7347 mM and 0.5565 mM, respectively),4 of 14both of
which were more active than TMP under the same conditions.

The effect
Figure1.1. The
Figure effect of
of target
targetcompounds
compounds on on
platelet aggregation
platelet in vitro.
aggregation (A) The
in vitro. ICThe
(A) 50 values
IC50 of
values of
target compounds against 10 µmol · L −−11 ADP-induced rabbit platelet aggregation in vitro; (B) The
target compounds against 10 µmol·L ADP-induced rabbit platelet aggregation in vitro; (B) The IC50
IC50 values
values of target
of target compoundsagainst
compounds against 1 mmol ·L−−1
mmol·L 1 AA-induced rabbit platelet aggregation in vitro
AA-induced rabbit platelet aggregation in vitro
(mean±±SD,
(mean SD,nn==5,5,**pp << 0.05, ** pp << 0.01
0.05, ** 0.01vs. vs.TMP).
TMP).

Firstly, we connected 6 with Aspirin and the NO donor to generate compound 14a.
Firstly, we connected 6 with Aspirin and the NO donor to generate compound 14a.
We expected that the compound could be hydrolyzed in vivo by esterases to release ligus-
We expected
trazine, Aspirin that
andthe
NO,compound
which would could be hydrolyzed
enhance the inhibitory in effects
vivo by onesterases to release ligus-
platelet aggregation
trazine, Aspirin and
due to synergism. To NO,
probewhich would
the effect enhance
of the the inhibitory
substitution position oneffects onwe
activity, platelet
designed aggrega-
tion
14bdue to synergism.
and 14c. In addition,To an probe the was
ether bond effect of thetosubstitution
applied connect the NO position on activity,
donor with aromaticwe de-
signed
acid to14b and 14c.
enhance In addition,
the stability an ether bond
and liposolubility of was applied
a fragment ofto
theconnect
NO donor.the NOThus, donor
we with
designed the second series of compounds 15a–15c. After these
aromatic acid to enhance the stability and liposolubility of a fragment of the NO compounds were tested fordonor.
activities, the best active compound of each type was selected, and
Thus, we designed the second series of compounds 15a–15c. After these compounds were we increased the length
of thefor
tested linker next to the
activities, theNObestdonor in the
active structure of
compound to each
searchtype
for an appropriate
was selected,length
and we of increased
the
carbon chain. Finally, we synthesized and tested target compounds 14d, 14e, 15d and 15e.
the length of the linker next to the NO donor in the structure to search for an appropriate
Among compounds 14a–14e, 14a has the best activity in ADP-induced and AA-
length of the carbon chain. Finally, we synthesized and tested target compounds 14d, 14e,
induced assays, with IC50 values of 0.7736 mM and 0.6236 mM. Compared with 13, which
15d and 15e. ligustrazine moiety and NO donor moiety in the structure, most of the
only contained
Amongthat
compounds compounds 14a–14e,
add in an Aspirin 14a has
fragment theabest
show activity
stronger in ADP-induced
inhibitory and AA-in-
effect in two assays,
duced
whichassays,
indicateswith
that IC values ofactivity
the50inhibitory 0.7736ismM and 0.6236
not strongly mM. due
increased Compared withaddi-
to the direct 13, which
tion of the NO donor, and the presence of the Aspirin fragment enhances
only contained ligustrazine moiety and NO donor moiety in the structure, most of the the activity against
platelet aggregation.
compounds that addThe in an activities
Aspirin offragment
compounds 14a–14e
show were significantly
a stronger inhibitoryaffected
effect inbytwo as-
says, which indicates that the inhibitory activity is not strongly increased duethe
the substitution position and length of the carbon chain of the NO-donor linker. On toone
the direct
hand, as with the substitution position of the hydroxyl group in Aspirin, the compound
addition of the NO donor, and the presence of the Aspirin fragment enhances the activity
with the NO-donor linker in the ortho position has the best activity in all assays. This
against platelet aggregation. The activities of compounds 14a–14e were significantly af-
further demonstrates the effect of Aspirin fragments on antiplatelet aggregation activity.
fected by
On the otherthe substitution position activities
hand, their inhibitory and length areof the carbon
markedly chainbyofthe
affected thelength
NO-donorof the linker.
On the one
carbon chainhand,
of theasNOwith
donor.theThe
substitution position
inhibitory activity of of the hydroxyl
compounds 14d and group in Aspirin, the
14e decreases
compound with the NO-donor linker
significantly when the linker length is increased. in the ortho position has the best activity in all as-
says. This further demonstrates the effect of Aspirin fragments on antiplatelet aggregation
activity. On the other hand, their inhibitory activities are markedly affected by the length
of the carbon chain of the NO donor. The inhibitory activity of compounds 14d and 14e
decreases significantly when the linker length is increased.
It is 15d that represents the best activity among compounds 15a–15e. The compounds
15a–15c were first synthesized and assayed to evaluate the optimal substitution position
Molecules 2023, 28, 3355 5 of 14

It is 15d that represents the best activity among compounds 15a–15e. The compounds
15a–15c were first synthesized and assayed to evaluate the optimal substitution position of
the NO-donor linker. The result showed that meta-substituted 15b displayed the optimal
inhibitory activity in both assays before we increased the length of the carbon chain of the
NO donor in the structure. However, the length of the carbon chain had an unremarkable
effect on the improvement in inhibitory activity according to the change in the IC50 values of
compounds 15d and 15e. Nevertheless, most of the compounds of 15a–15e displayed better
activity than compounds 14a–14e. As mentioned above, the preliminary structure-activity
relationship revealed that the compound derivatives that differed in the composition of
the active structure fragment, substituent position and length of the carbon chain of the
NO-donor linker showed varied activities against platelet aggregation.

2.3. Molecular Docking

The thromboxane A2 receptor (TBXA2R) plays a key role in physiological processes,
such as platelet aggregation and the contraction of vascular smooth muscle cells; thus it is
often used as an important target in the study of drugs for the treatment of cardiovascular
and cerebrovascular diseases, such as atherosclerosis and cerebral ischemia [20]. Based on
the characteristics of this receptor, this study docked with the designed compound at the
active site.
The results of docking efficiency (-CDOCKER_INTERACTION_ENERGY) were ba-
sically consistent with the results of the corresponding cell experiments, especially for
compounds 15a–15e (Table S2). It was found that the main forms of the interactions be-
tween TMP and proteins are Pi-alkyl and hydrophobic bonds of alkyl, and the interactions
between TMP and proteins only occur on the surface layer of the active site instead of in the
depths of the protein void, resulting in low docking efficiency (Figure 2A). In contrast, the
newly designed ligustrazine derivative increased the length of the molecule, allowing the
compound structure to penetrate deeper into the protein cavity at the active site and interact
with it. For example, the results of the interaction between 14a and TBXA2R showed that the
changes in the structure and volume of the compound promoted deeper binding between
14a and the protein cavity, and the types and number of non-bond interactions between 14a
and TBXA2R were more abundant (Figure 2B). By comparing the results of the interactions
between 14a, 14b and 14c with the receptor, instead of para-substituted 14c at the active
site showing an obvious linear structure extending inwardly, ortho-substituted compound
14a, at the active site, showed a larger number and types of beneficial interactions with the
receptor, which increased the interaction intensity. In the structure of compounds 15a–15e,
the benzene ring and pyrazine rings were relatively close together, but they had a greater
number of non-bond interactions with the acceptor, which seemed to suggest that the
existence of a double bond between aryl groups in the structure of compounds 14a–14e
was not appropriate (Figure 2C). Furthermore, a 2D interaction diagram analysis further
showed that there was less interaction between the active site of the receptor and the carbon
chain of the NO donor, which pointed out that the length of the carbon chain could not
have a positive effect on the interaction between the compound and the receptor.
Based on the above results, we speculated that the mode of action of the compounds
might be to inhibit AA-induced platelet aggregation by acting on the active site of TBXA2R.
The Aspirin structure contributed more to the binding between the compounds and
TBXA2R, while the length of the linking carbon chain produced a greater effect on the stabil-
ity of the NO donor and release of NO. In addition, it is likely that various compositions of
fragment molecules and NO-donor linkers may have different effects on the liposolubility
and metabolism of target compounds, leading to different inhibitory activities.
 Molecules 2023, 28, x FOR PEER REVIEW 6 of 14
Molecules 2023, 28, 3355 6 of 14

Figure2.2.(A)
Figure (A)TMP,
TMP,(B)
(B)14a
14a and
and (C)
(C) 15d
15d and
and interactions
interactions with
with TBXA2R
TBXA2R (PDB
(PDB 6IIV).
6IIV).

Based on the above results, we speculated that the mode of action of the compounds
might be to inhibit AA-induced platelet aggregation by acting on the active site of
Molecules 2023, 28, 3355 7 of 14

3. Materials and Methods

3.1. Chemistry
All chemicals were of analytical reagent grade, and all solvents were of reagent grade.
The reactions were monitored by thin-layer chromatography (TLC) (Qingdao Haiyang
Chemical Co., Qingdao, China). The solutions containing the products were concentrated
with a rotary evaporator. Flash column chromatography was performed in a column
packed with 200–300 mesh silica gel. TLC was performed on silica gel GF254-coated glass
sheets, and the spots were visualized under UV light (254 nm). NMR spectra were recorded
with the Agilent DD2 600 MHz NMR spectrometer (Agilent Technologies Inc., Santa Clara,
CA, USA) in CDCl3 containing 0.03% v/v tetramethylsilane (TMS) as the internal standard.
The HRMS was recorded on a Xevo G2-XS QTOF instrument (Waters, MA, USA).

3.1.1. General Procedure for the Preparation of 1

NaOH (8.0 g, 200 mmol) and ligustrazine hydrochloride (41.8 g, 200 mmol) were
dissolved in 300 mL and 100 mL of water at 0 ◦ C to prepare solutions, respectively. To the
stirred solution of ligustrazine hydrochloride was added slowly the NaOH solution. After
stirring for one hour, water was removed under reduced pressure, and the product was
collected by filtration and dried by a standard method to obtain ligustrazine trihydrate.
Then, a solution of ligustrazine trihydrate (20.4 g, 107 mmol) in glacial acetic acid (30 mL)
was added to the 30% H2 O2 aqueous solution (12.1 mL, 107 mmol). The reaction mixture
was stirred at 90 ◦ C for 2 h before another portion of the 30% H2 O2 aqueous solution
(12.1 mL, 107 mmol) was added and stirred at 90 ◦ C for another 2 h. The resultant mixture
was cooled to room temperature, alkalized to pH = 10 with 50% sodium hydroxide and
extracted with dichloromethane (150 mL, 50 mL × 3). The organic layer was combined and
dried with anhydrous sodium sulfate for 8 h and evaporated in vacuo to afford ligustrazine
mono-N-oxide (1).

3.1.2. General Procedure for the Preparation of 3

Compound 1 (13.8 g, 90.6 mmol) was treated with acetic anhydride (17.1 mL, 181.2 mmol)
at 130 ◦ C for 2.5 h. The mixture was added in 20% NaOH (aq, 100 mL) and stirred at room
temperature overnight. The resultant mixture was extracted with dichloromethane (160 mL,
40 mL × 4). The extract was dried with anhydrous sodium sulfate for 8 h and evaporated
in vacuo to obtain the crude product. The crude product was recrystallized from a mixed
solvent of petroleum ether (PE) and ethyl acetate (EA) (PE/EA = 10/1, v/v) to obtain 3 as a
yellow solid (5.5 g), an overall yield of 33.7%.

3.1.3. General Procedure for the Preparation of 4

To a solution of 3 (6.09 g, 40 mmol) in anhydrous ethanol (20 mL), active MnO2 was
added (20.8 g, 240 mmol). After heating to reflux for 6 h, the reaction mixture was filtered
through a sintered glass funnel layered with a layer of diatomite, and the filtrate was
evaporated in vacuo to obtain 3,5,6-trimethylpyrazine-2-carbaldehyde (4) as a yellow solid
(5.67 g).

3.1.4. General Procedure for the Preparation of 5

To a solution of compound 4 (5.5 g, 36.6 mmol) in toluene (50 mL), NaH (1.76 g,
73.2 mmol) was added carefully in an ice bath, and after 0.5 h, triethyl phosphonoacetate
(9.0 g, 40.3 mmol) was added dropwise, and the mixture was stirred at room temperature
overnight. The reaction solution was diluted by adding EtOAc (50 mL), then washed with
saturated brine (50 mL × 3), and the combined extract was dried over anhydrous sodium
sulfate for 8 h and concentrated in vacuo. The crude product was purified by column
chromatography (PE/EA = 8/1, v/v) to obtain 5 as a pale-yellow solid (6.45 g).
Molecules 2023, 28, 3355 8 of 14

3.1.5. General Procedure for the Preparation of 6

Compound 5 (6.45 g, 29.3 mmol) was dissolved in a mixed solvent of tetrahydrofuran
(THF) and water (THF/H2 O = 2/1, v/v, 30 mL), and then sodium hydroxide (2.35 g,
58.6 mmol) was slowly added, and the mixture was stirred at room temperature for 4 h.
The reaction mixture was adjusted with 2 mol/L of hydrochloric acid to pH = 4, followed
by the addition of sodium chloride solid until it dissolved more. Next, the mixture was
extracted with EtOAc (90 mL, 30 mL × 3), and the combined organic phase was dried over
anhydrous sodium sulfate for 8 h and evaporated in vacuo to obtain 6 as a white solid
(5.5 g).

3.1.6. General Procedure for the Preparation of 7

2-Bromoethanol (1.25 g, 10 mmol) was dissolved in acetonitrile (50 mL), and AgNO3
(2.55 g, 15 mmol) was added to the reaction solution, which was then stirred under darkness
at 70 ◦ C for 3 h. After cooling, the reaction mixture was filtered to remove the solid
product, and the filtrate was concentrated in vacuo. The concentrate was stirred for 10 min
after adding 30 mL EtOAc, then filtered and concentrated again to obtain 7 (1.0 g) as a
yellow liquid.

3.1.7. General Procedure for the Preparation of 8a–e

The corresponding dibromoethane (7.5 mmol,1.5 eq) and triethylamine (7.5 mmol,
1.5 eq) were added to a solution of hydroxybenzoic acid (0.69 g, 5 mmol) in acetone (20 mL).
After heating to reflux for 6 h under nitrogen, the reaction mixture was cooled to room
temperature and evaporated in vacuo. The residue was stirred for 5 min after adding 30 mL
of EtOAc and then filtered and concentrated. The concentrate was purified by column
chromatography (PE/EA = 10/1, 8/1, v/v) to obtain 8a–e: 8a, colorless oil, PE/EA = 8/1,
yield 22%; 8b, colorless oil, PE/EA = 8/1, yield 27%; 8c, white solid, PE/EA = 8/1, yield
29%; 8d, colorless oil, PE/EA = 10/1, yield 61%; 8e, colorless oil, PE/EA = 10/1, yield 60%.

3.1.8. General Procedure for the Preparation of 9a–e

DCC (0.23 g, 1.1 mmol) and 6 (0.19 g, 1.0 mmol) were dissolved in anhydrous
dichloromethane (20 mL), and a catalytic amount of DMAP was added to the reaction
solution, which was then stirred for about 15 min. Next, 8a–e (1.1 mmol,1.1 eq) was added
to the solution and stirred for 4 h at room temperature. The resultant mixture was filtered,
and the filtrate was concentrated in vacuo. The concentrate was purified by column chro-
matography (PE/EA = 5/1, 4/1, v/v) to obtain 9a–e: 9a, white powder, PE/EA = 5/1, yield
67%; 9b, white powder, PE/EA = 5/1, yield 70%; 9c, white powder, PE/EA = 5/1, yield
70%; 9d, yellow solid, PE/EA = 5/1, yield 72%; 9e, white solid, PE/EA = 5/1, yield 93%.

3.1.9. General Procedure for the Preparation of 10a–e

To a solution of hydroxybenzaldehyde (1.22 g, 10 mmol) in acetone (20 mL), the
corresponding brominated alcohol (20 mmol, 2.0 eq) and anhydrous K2 CO3 (20 mmol,
2.0 eq) were added. After heating to reflux for 6 h under nitrogen, the reaction mixture
was cooled to room temperature and concentrated in vacuo. The concentrate was purified
by column chromatography (PE/EA = 4/1, 2/1, v/v) to obtain 10a–e: 10a, yellow oil,
PE/EA = 4/1–2/1, yield 38%; 10b, cream oil, PE/EA = 4/1, 2/1, yield 29%; 10c, mauve
oil, PE/EA = 3/1, yield 67%; 10d, yellow oil, PE/EA = 4/1, yield 35%; 10e, yellow oil,
PE/EA = 4/1, yield 58%.

3.1.10. General Procedure for the Preparation of 11a–e

Compounds 10a–e (1.0 eq) and a small amount of carbamide were dissolved in glacial
acetic acid (5 mL), and mixtures of Fuming HNO3 (5.0 eq) and acetic anhydride (5 mL)
were slowly added dropwise at −5 ◦ C. The reaction solution was stirred at −5 ◦ C for
0.5 h and then kept at room temperature for 2 h. The resultant solution was poured into
ice water, then adjusted to pH = 7 with the saturated NaHCO3 solution and extracted
Molecules 2023, 28, 3355 9 of 14

with EtOAc (60 mL, 20 mL × 3). The combined organic layer was washed with brine,
dried over anhydrous sodium sulfate for 8 h and concentrated in vacuo. The concentrate
was purified by column chromatography (PE/EA = 4/1, 3/1, v/v) to obtain 11a–e: 11a,
yellow oil, PE/EA = 4/1, yield 66%; 11b, yellow oil, PE/EA = 4/1, yield 63%; 11c, yellow
oil, PE/EA = 3/1, yield 60%; 11d, yellow oil, PE/EA = 4/1, yield 61%; 11e, yellow oil,
PE/EA = 4/1, yield 63%.

3.1.11. General Procedure for the Preparation of 12a–e

KMnO4 (1.5 eq) was added to a solution of 11a–e (1.0 eq) in acetone (20 mL) and
heated to reflux for 2 h. The reaction mixture was added to the saturated NaHSO3 solution
until its red faded completely. Then, the pH of the solution was adjusted to 5 with 2 mol/L
of hydrochloric acid, and after being filtered through a sintered glass funnel layered with a
layer of diatomite, the filtrate was extracted with EtOAc (60 mL, 20 mL × 3). The combined
organic extracts were washed with brine, dried over anhydrous sodium sulfate for 8 h and
evaporated in vacuo to afford 12a–e: 12a, yellow oil, yield 82%; 12b, yellow solid, yield
88%; 12c, white solid, yield 90%; 12d, yellow oil, yield 86%; 12e, yellow oil, yield 85%.

3.1.12. 2-(Nitrooxy)ethyl (E)-3-(3,5,6-Trimethylpyrazin-2-yl)acrylate (13)

Both 6 (0.74 g, 3.8 mmol) and DCC (0.79 g, 3.8 mmol) were dissolved in anhydrous
dichloromethane (30 mL), and a catalytic amount of DMAP was added to the reaction
solution, which was then stirred for about 15 min. Next, compound 7 (0.38 g, 3.5 mmol)
was added to the solution and stirred for 6 h at room temperature. The resultant mixture
was filtered, and the filtrate was concentrated in vacuo. The concentrate was stirred for
15 min after adding 20 mL EtOAc, then filtered and concentrated again, and was purified
by column chromatography (PE/EA = 5/1, v/v) to obtain target compound 13 (0.40 g),
with an overall yield of 41%. White solid, 1 H NMR (600 MHz, Chloroform-d): δ 7.88 (d,
1H, J = 15.3 Hz, ArCH = CH), 7.06 (d, 1H, J = 15.3 Hz, ArCH = CH), 4.79–4.72 (m, 2H,
CH2 CH2 NO3 ), 4.55–4.48 (m, 2H, CH2 CH2 NO3 ), 2.62 (s, 3H, ArCH3 ), 2.52 (d, 6H, J = 4.6 Hz,
ArCH3 ); 13 C NMR (151 MHz, CDCl3 ) δ 166.28, 153.01, 150.02, 149.12, 142.25, 140.06, 121.87,
77.20, 76.99, 76.77, 70.44, 60.36, 22.02, 21.69, 20.66; HRMS (ESI): m/z: 281.1112 calc. for
C12 H15 N3 O5 [M + H]+ , found 281.1109, ppm error −1.1.

3.1.13. General Procedure for the Preparation of 14a–14e

9a–e (1.0 eq) was dissolved in acetonitrile (20 mL), and AgNO3 (1.5 eq) was added
to the reaction solution, which was then stirred under darkness at 70 ◦ C for 3 h. After
cooling, the reaction mixture was filtered to remove the solid product, and the filtrate
was concentrated in vacuo. The concentrate was stirred for 10 min after adding 30 mL
of EtOAc, then filtered and concentrated again, and purified by column chromatography
(PE/EA = 4/1, 2/1, v/v) or TLC (DCM/MeOH = 50/1, v/v) to obtain target compounds
14a–14e.
2-(nitrooxy)ethyl (E)-2-((3-(3,5,6-trimethylpyrazin-2-yl)acryloyl)oxy)benzoate (14a).
Cream solid, PE/EA = 2/1, yield 37%; 1 H NMR (600 MHz, Chloroform-d): δ 8.08 (d, 1H,
J = 6.4 Hz, 6-ArH), 8.05 (d, 1H, J = 15.6 Hz, ArCH = CH), 7.60 (t, 1H, J = 7.8 Hz, 4-ArH),
7.36 (t,1H, J = 7.6 Hz, 5-ArH), 7.31 (d, 1H, J = 15.3 Hz, ArCH = CH), 7.19 (d, 1H, J = 8.0 Hz,
3-ArH), 4.38 (t, 2H, J = 4.5 Hz, CH2 CH2 NO3 ), 3.84 (t, 2H, J = 4.5 Hz, CH2 CH2 NO3 ), 2.66 (s,
3H, ArCH3 ), 2.56 (d, 6H, J = 5.9 Hz, ArCH3 ); 13 C NMR (151 MHz, CDCl3 ) δ 165.28, 164.98,
152.75, 150.56, 150.22, 148.86, 142.79, 140.50, 133.99, 132.03, 126.19, 123.78, 123.43, 122.71,
77.22, 77.01, 76.80, 66.93, 60.71, 21.63, 21.61, 20.29; HRMS (ESI): m/z: 402.1301 calc. for
C19 H20 N3 O7 [M + H]+ , found 402.1291, ppm error −2.5.
2-(nitrooxy)ethyl (E)-3-((3-(3,5,6-trimethylpyrazin-2-yl)acryloyl)oxy)benzoate (14b).
Yellow solid, PE/EA = 2/1, yield 64%; 1 H NMR (600 MHz, Chloroform-d) δ 8.03 (d, 1H,
J = 15.2 Hz, ArCH = CH), 7.96 (d, 1H, J = 7.8 Hz, 6-ArH), 7.86 (s, 1H, 2-ArH), 7.49 (m, 1H,
J = 7.9 Hz, 5-ArH), 7.39 (d, 1H, J = 8.1 Hz, 4- ArH), 7.24 (d, 1H, J = 15.3 Hz, ArCH = CH),
4.47 (t, 2H, J = 4.6 Hz, CH2 CH2 NO3 ), 3.96 (t, 2H, J = 4.6 Hz, CH2 CH2 NO3 ), 2.64 (s, 3H,
Molecules 2023, 28, 3355 10 of 14

ArCH3 ), 2.55 (s, 6H, ArCH3 ); 13 C NMR (151 MHz, CDCl3 ) δ 165.93, 164.95, 153.15, 150.68,
150.26, 149.17, 142.28, 140.95, 131.41, 129.49, 127.12, 126.51, 122.89, 121.88, 77.22, 77.01, 76.80,
66.90, 61.19, 21.95, 21.71, 20.58; HRMS (ESI): m/z: 402.1301 calc. for C19 H20 N3 O7 [M + H]+ ,
found 402.1300, ppm error −0.2.
2-(nitrooxy)ethyl (E)-4-((3-(3,5,6-trimethylpyrazin-2-yl)acryloyl)oxy)benzoate (14c).
Yellow solid, PE/EA = 2/1, yield 50%; 1 H NMR (600 MHz, Chloroform-d): δ 8.12 (d, 2H,
J = 8.5 Hz, 2,6-ArH), 8.03 (d, 1H, J = 15.2 Hz, ArCH = CH), 7.31–7.24 (m, 3H, 3,5-ArH and
ArCH = CH), 4.48 (t, 2H, J = 4.7 Hz, CH2 CH2 NO3 ), 3.97 (t, 2H, J = 4.6 Hz, CH2 CH2 NO3 ),
2.67 (s, 3H, ArCH3 ), 2.58 (d, 6H, J = 7.2 Hz, ArCH3 ); 13 C NMR (151 MHz, CDCl3 ) δ
166.11, 164.47, 154.54, 152.61, 150.83, 148.59, 142.77, 140.60, 131.29, 127.43, 122.34, 121.63,
77.21, 77.00, 76.79, 66.77, 61.33, 21.71, 21.52, 20.12; HRMS (ESI): m/z: 402.1301 calc. for
C19 H20 N3 O7 [M + H]+ , found 402.1299, ppm error −0.5.
4-(nitrooxy)butyl (E)-2-((3-(3,5,6-trimethylpyrazin-2-yl)acryloyl)oxy)benzoate (14d).
Cream solid, PE/EA = 4/1, yield 39%; 1 H NMR (600 MHz, Chloroform-d) δ 8.06 (d, 1H,
J = 14.7 Hz, ArCH = CH), 8.04 (d, 1H, J = 7.9 Hz, 6-ArH), 7.60 (t, 1H, J = 7.8 Hz, 4-ArH),
7.36 (t, 1H, J = 7.7 Hz, 5-ArH), 7.32 (d, 1H, J = 15.2 Hz, ArCH = CH), 7.19 (d, 1H, J = 8.1 Hz,
3-ArH), 4.43 (t, 2H, J = 5.9 Hz, CH2 NO3 ), 4.30 (t, 2H, J = 5.6 Hz, COOCH2 ), 2.65 (s, 3H,
ArCH3 ), 2.55 (d, 6H, J = 5.6 Hz, ArCH3 ), 1.87–1.77 (m, 4H, CH2 CH2 CH2 CH2 ); 13 C NMR
(151 MHz, CDCl3 ) δ 165.17, 164.62, 153.10, 150.39, 150.23, 149.14, 142.28, 140.79, 133.91,
131.78, 126.11, 123.84, 123.52, 122.00, 77.22, 77.01, 76.79, 72.53, 64.18, 24.99, 23.62, 21.97,
21.70, 20.58; HRMS (ESI): m/z: 430.1614 calc. for C21 H24 N3 O7 [M + H]+ , found 430.1617,
ppm error 0.7.
6-(nitrooxy)hexyl (E)-2-((3-(3,5,6-trimethylpyrazin-2-yl)acryloyl)oxy)benzoate (14e).
Yellow oil, DCM/MeOH = 50/1, yield 59%; 1 H NMR (600 MHz, Chloroform-d) δ 8.01 (d, 1H,
J = 15.3 Hz, ArCH = CH), 8.00 (d, 1H, J = 8.5 Hz, 6-ArH), 7.55 (t, 1H, J = 7.7 Hz, 4-ArH), 7.31 (t,
1H, J = 6.8 Hz, 5-ArH), 7.29 (d, 1H, J = 15.4 Hz, ArCH = CH), 7.15 (d, 1H, J = 8.1 Hz, 3-ArH),
4.34 (t, 2H, J = 6.6 Hz, CH2 NO3 ), 4.22 (t, 2H, J = 6.6 Hz, COOCH2 ), 2.61 (s, 3H, ArCH3 ),
2.51 (d, 6H, J = 4.1 Hz, ArCH3 ), 1.65 (td, 4H, J = 14.5, 7.6 Hz, CH2 CH2 CH2 CH2 CH2 CH2 ),
1.37 (td, 4H, J = 11.7, 10.4, 6.8 Hz, CH2 CH2 CH2 CH2 CH2 CH2 ); 13 C NMR (151 MHz, CDCl3 )
δ 165.15, 164.69, 153.08, 150.38, 150.08, 149.15, 142.27, 140.63, 133.71, 133.68, 131.78, 131.74,
126.04, 126.02, 123.81, 123.79, 123.77, 122.12, 122.09, 77.27, 77.06, 76.85, 73.06, 65.07, 64.92,
28.44, 28.39, 26.55, 25.62, 25.54, 25.29, 25.17, 22.03, 22.01, 21.73, 21.69, 20.67, 20.65; HRMS
(ESI): m/z: 458.1927 calc. for C23 H28 N3 O7 [M + H]+ , found 458.1927, ppm error 0.0.

3.1.14. General Procedure for the Preparation of 15a–15e

Compound 12a–e (1.2 mmol, 1.2 eq) and DCC (1.2 mmol,1.2 eq) were dissolved in
anhydrous dichloromethane (20 mL), and a catalytic amount of DMAP was added to the
reaction solution, which was then stirred for about 15 min. Next, 3 (1.0 mmol,1.0 eq) was
added to the solution and stirred for 6 h at room temperature. The resultant mixture was
filtered, and the filtrate was concentrated in vacuo. The concentrate was purified by TLC
(PE/EA/EtOH = 30/10/1, v/v/v) to obtain the target compounds 15a–15e.
(3,5,6-trimethylpyrazin-2-yl)methyl 2-(2-(nitrooxy)ethoxy)benzoate (15a). Yellow solid,
43% yield; 1 H NMR (600 MHz, Chloroform-d): δ 7.85 (d, 1H, J = 7.8 Hz, 6-ArH), 7.47 (t,
J = 7.8 Hz, 1H, 4-ArH), 7.04 (t, J = 7.6 Hz, 1H, 5-ArH), 6.94 (d, 1H, J = 8.3 Hz, 3-ArH), 5.42 (s,
2H, ArCH2 ), 4.72 (t, 2H, J = 4.7 Hz, CH2 CH2 NO3 ), 4.28 (t, 2H, J = 4.7 Hz, CH2 CH2 NO3 ),
2.59 (s, 3H, ArCH3 ), 2.52 (d, 6H, J = 6.9 Hz, ArCH3 ); 13 C NMR (151 MHz, CDCl3 ) δ 165.54,
157.63, 151.38, 149.24, 148.97, 144.81, 133.69, 132.09, 121.60, 120.81, 114.28, 77.24, 77.02, 76.81,
70.75, 65.70, 65.57, 21.61, 21.36, 20.52; HRMS (ESI): m/z: 362.1352 calc. for C17 H20 N3 O6
[M + H]+ , found 362.1361, ppm error 2.5.
(3,5,6-trimethylpyrazin-2-yl)methyl 3-(2-(nitrooxy)ethoxy)benzoate (15b). Cream solid,
54% yield; 1 H NMR (600 MHz, Chloroform-d): δ 7.69 (dd, 1H, J = 7.7, 1.4 Hz, 6-ArH), 7.55 (s,
1H, 2-ArH), 7.35 (t, 1H, J = 7.9 Hz, 5-ArH), 7.11 (dd, 1H, J = 8.3, 2.9 Hz, 4-ArH), 5.44 (s, 2H,
ArCH2 ), 4.82 (t, 2H, J = 4.6 Hz, CH2 CH2 NO3 ), 4.28 (t, 2H, J = 4.5 Hz, CH2 CH2 NO3 ), 2.59 (s,
3H, ArCH3 ), 2.52 (d, 6H, J = 9.8 Hz, ArCH3 ); 13 C NMR (151 MHz, CDCl3 ) δ 165.81, 157.90,
Molecules 2023, 28, 3355 11 of 14

151.47, 149.26, 149.02, 144.68, 131.19, 129.65, 123.06, 120.22, 114.69, 77.24, 77.03, 76.82, 70.73,
65.93, 64.22, 21.63, 21.37, 20.54; HRMS (ESI): m/z: 362.1352 calc. for C17 H20 N3 O6 [M + H]+ ,
found 362.1353, ppm error 0.3.
(3,5,6-trimethylpyrazin-2-yl)methyl 4-(2-(nitrooxy)ethoxy)benzoate (15c). White solid,
45% yield; 1 H NMR (600 MHz, Chloroform-d): δ 8.01 (d, 2H, J = 8.5 Hz, 2,6-ArH), 6.90 (d,
2H, J = 8.3 Hz, 3,5-ArH), 5.42 (s, 2H, ArCH2 ), 4.83 (t, 2H, J = 4.5 Hz, CH2 CH2 NO3 ), 4.30 (t,
2H, J = 4.5 Hz, CH2 CH2 NO3 ), 2.58 (s, 3H, ArCH3 ), 2.52 (d, 6H, J = 8.3 Hz, ArCH3 ); 13 C
NMR (151 MHz, CDCl3 ) δ 165.69, 161.72, 151.33, 149.28, 148.93, 144.93, 131.86, 131.80,
123.06, 114.15, 114.10, 77.26, 77.05, 76.84, 70.57, 65.65, 64.07, 21.63, 21.38, 20.55; HRMS (ESI):
m/z: 362.1352 calc. for C17 H20 N3 O6 [M + H]+ , found 362.1351, ppm error −0.3.
(3,5,6-trimethylpyrazin-2-yl)methyl 3-(4-(nitrooxy)butoxy)benzoate (15d). Colorless
oil, 54% yield; 1 H NMR (600 MHz, Chloroform-d): δ 7.59 (dd, 1H, J = 7.7, 1.3 Hz, 6-ArH),
7.49 (s, 1H, 2-ArH), 7.28 (t, J = 8.0 Hz, 1H, 5-ArH), 7.04 (dd, 1H, J = 8.3, 2.6 Hz, 4-ArH),
5.39 (s, 2H, ArCH2 ), 4.49 (t, 2H, J = 6.1 Hz, CH2 NO3 ), 3.98 (t, 2H, J = 5.6 Hz, OCH2 ),
2.54 (s, 3H, ArCH3 ), 2.48 (d, 6H, J = 9.2 Hz, ArCH3 ), 1.88 (ddt, 4H, J = 10.7, 5.8, 2.2 Hz,
CH2 CH2 CH2 CH2 ); 13 C NMR (151 MHz, CDCl3 ) δ 166.03, 158.69, 151.42, 149.31, 148.99,
144.79, 131.06, 129.48, 122.33, 119.98, 114.77, 77.22, 77.01, 76.79, 72.79, 67.13, 65.92, 25.46,
23.80, 21.65, 21.40, 20.57; HRMS (ESI): m/z: 390.1665 calc. for C19 H24 N3 O6 [M + H]+ , found
390.1668, ppm error 0.8.
(3,5,6-trimethylpyrazin-2-yl)methyl 3-((6-(nitrooxy)hexyl)oxy)benzoate (15e). Color-
less oil, 49% yield; 1 H NMR (600 MHz, Chloroform-d) δ 7.58 (dd, 1H, J = 7.7, 1.4 Hz, 6-ArH),
7.50 (s, 1H, 2-ArH), 7.27 (t, 1H, J = 8.0 Hz, 5-ArH), 7.04 (dd, 1H, J = 8.3, 2.6 Hz, 4-ArH),
5.39 (s, 2H, ArCH2 ), 4.41 (t, 2H, J = 6.7 Hz, CH2 NO3 ), 3.94 (t, 2H, J = 6.3 Hz, OCH2 ), 2.54 (s,
3H, ArCH3 ), 2.47 (d, 6H, J = 8.9 Hz, ArCH3 ), 1.79–1.67 (m, 4H, CH2 CH2 CH2 CH2 CH2 CH2 ),
1.51–1.39 (m, 4H, CH2 CH2 CH2 CH2 CH2 CH2 ); 13 C NMR (151 MHz, CDCl3 ) δ 166.11, 158.96,
151.40, 149.33, 148.97, 144.83, 130.99, 129.40, 122.07, 120.00, 114.84, 77.21, 77.00, 76.79, 73.16,
67.81, 65.91, 28.92, 26.69, 25.62, 25.42, 21.65, 21.40, 20.58; HRMS (ESI): m/z: 418.1978 calc.
for C21 H28 N3 O6 [M + H]+ , found 418.1978, ppm error 0.0.

3.1.15. Data Analysis of NMR Spectra

NMR spectra were analyzed using the software MestReNova 14.0 after confirming
that the measured molecular mass in the HRMS of individual compounds matched their
calculated value. The 1 H NMR spectral peak reports of the compounds were exported
after processing for peak picking, integration and multiplets analysis using the software
MestReNova 14.0. Individual peaks’ data were analyzed, and the corresponding hydrogen
atom was found in the compound structure. The peaks in 13 C NMR spectra were also
picked and reported using this software, and the data of the characteristic carbon peaks
(C=O, – CH3 ) were analyzed.

3.2. In Vitro Antiplatelet Aggregation Assay Induced by ADP and AA

New Zealand white (NZW) rabbits were purchased from the Qinglongshan Animal
Breeding Center. Our experimental protocols were approved by the Wuhan University of
Science and Technology Institutional Animal Care and Use Committee (IACUC; 202033)
and carried out in accordance with the EU Directive 2010/63/EU for animal experiments.
Blood samples were withdrawn from the rabbit carotid artery and infused into a centrifuge
tube containing 1/10 of the volume of 3.8% sodium citrate solution, followed by mixing.
After centrifugation at 112× g for 15 min at room temperature to obtain the supernatants
as platelet-rich plasma (PRP), the remaining blood was further centrifuged at 1008× g
for another 15 min to obtain platelet-poor plasma (PPP). The PPP was used to adjust the
PRP concentration to 1 × 108 mL−1 . The aggregation rate of the individual compounds
was measured by Born’s turbidimetric method at 37 ◦ C using a Platelet Aggregometer
(LBY-NJ4A Platelet-Aggregometer, Beijing Precil Instrument Co., Beijing).
Briefly, the PRP (260 µL) was preincubated in duplicate for 5 min at 37 ◦ C with the
vehicle, individual compounds or reference drugs (TMP) at the same concentrations (0.1,
Molecules 2023, 28, 3355 12 of 14

0.2, 0.4, 0.8, 1.6, 3.2 mM) and the platelet aggregation in the individual PPR samples was
induced by adding 30 µL of ADP (final concentration of 10 µM) or AA (final concentration
of 1 mM), followed by the recording of light transmission at maximal aggregation within
5 min. The rabbit PRP treated with vehicles was used as a positive control. The inhibition
rate of the tested individual compounds was calculated as the following formula: inhibition
rate (%) = [(1 − (the platelet aggregation in samples with the tested compound)/(the
platelet aggregation in control samples)] × 100. The IC50 values of the tested compounds
were determined by nonlinear regression analysis using GraphPad Prism 9.0. Data are
expressed as the mean ± SD of each group (n = 5) and are analyzed by one-way analysis of
variance (ANOVA) with Tukey’s test.

3.3. Molecular Docking

Molecular docking calculations were performed by Discovery Studio software using
the TBXA2R. First, we downloaded the protein’s 3D structure PDB format file through
the RSCB PDB database (PDB ID: 6IIV) and used hydrogenation and dehydration of the
Macromolecules > Prepare Protein module in Discovery Studio software. Further, 6IIV is
defined as a receptor through the Receptor-Ligand Interactions module. Set (X = 24.749525,
Y = 162.147302, Z = 144.741603, Radius = 10.5000) as the active docking site [21]. The
Chemoffice software was used to draw the structures of active compounds, and after
importing them into the Discovery Studio software, the Small Molecules > Prepare or
Filter Ligands module was used to prepare the compounds. After the preparation of
the receptors and ligands, precise molecular docking between compounds and proteins
was completed through the Receptor-Ligand Interactions > Dock Ligands (CDOCKER)
module [22]. Further, -CDOCKER_INTERACTION_ENERGY TOP#1 was used as the
screening condition to obtain the best results of the interaction between each compound
and protein.

4. Conclusions
In conclusion, we designed and synthesized a series of novel nitric oxide-donating
ligustrazine derivatives. All the derivatives exhibited inhibitory activities on platelet aggre-
gation induced by ADP or AA in vitro. Compounds 14a and 15d displayed significantly
better activity than ligustrazine in both assays. Preliminary in vitro studies have shown
that it was a viable strategy for improving the effects due to the addition of the NO donor in
the structure. Additionally, we studied docking with the novel derivatives at the active site
of TBXA2R and discussed the structure-activity relationship, which will provide a reference
for the research of this class of compounds. As a result, our present studies suggested
that the novel NO-donating ligustrazine derivatives 14a and 15d deserve further study as
potent antiplatelet aggregation agents.

Supplementary Materials: The following supporting information can be downloaded at: https:
//www.mdpi.com/article/10.3390/molecules28083355/s1, Table S1: The IC50 values of target com-
pounds against platelet aggregation in vitro induced by ADP or AA, Table S2: Docking energies of
ligustrazine (TMP) and all new compounds (13, 14a–14e, 15a–15e) with TBXA2R. Binding modes of
all the compounds at the active site of TBXA2R. 1 H-NMR, 13 C-NMR and HRMS spectroscopy of all
new compounds.
Author Contributions: Conceptualization, H.-X.L. and Y.Z.; methodology, H.-X.L.; software, J.-H.T.
and H.-Y.L.; validation, H.-X.L. and Y.Z.; formal analysis, H.-X.L. and J.-H.T.; investigation, H.-
X.L., H.-Y.L. and X.W.; resources, H.-X.L. and Y.Z.; data curation, H.-X.L., J.-H.T., H.-Y.L. and X.W.;
writing—original draft preparation, H.-X.L. and J.-H.T.; writing—review and editing, H.-X.L. and Y.Z.;
visualization, H.-X.L. and J.-H.T.; supervision, Y.Z.; project administration, Y.Z.; funding acquisition,
Y.Z. All authors have read and agreed to the published version of the manuscript.
Molecules 2023, 28, 3355 13 of 14

Funding: This work was supported by the National Natural Science Foundation of China (Grant
NO. 82003593), the Natural Science Foundation of Hubei Province of China (Grant NO. 2019CFB104)
and the College Students’ Innovative Entrepreneurial Training Plan Program of Hubei Province
(S201910488066).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data used to support the findings of this study are included within
the article and Supplementary Materials.
Acknowledgments: The authors are grateful to the BioRN Life Science Company for conducting
antiplatelet aggregation assays and to China Pharmaceutical University for providing access to
Discovery Studio 2019.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Not applicable.

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